Urinary tract infections (UTIs) caused by extraintestinal pathogenic E. coli (ExPEC) account for nearly 1.3 million emergency room visits and 245,000 hospitalizations per year for women in the U.S. alone. Approximately half of all women will experience a symptomatic UTI in their lifetime, and 25% will experience a recurrence within 6 to 12 months. While several attempts have been made to design a vaccine to address this important medical need, no vaccines are currently available. The goal of this proposal is to develop a broad- spectrum, multivalent subunit vaccine based on bacterial iron receptor proteins (IRPs). Iron is an essential nutrient for bacteria, and IRPs make excellent vaccine targets because they are highly conserved and are induced during infection in response to the low availability of iron in the host. Our innovative vaccine production strategy for bacteri mimics iron restriction through use of an iron chelator to enrich the content of these proteins on the surface of the cell, whereupon they can be purified through a proprietary platform process to create a subunit vaccine. Protective immunity to a pathogen such as ExPEC likely requires an optimal immune response at mucosal surfaces. Bacterial IRPs are attractive targets but present a significant challenge for vaccine development due to their insolubility and the low efficiency with which these antigens can be delivered across the mucosal epithelium to the underlying lymphoid tissue. The current proposal will address these issues by combining a novel IRP antigen composition with several innovative CpG delivery systems in order to maximize mucosal immune responses to ExPEC and provide protection against UTI. In Aim 1, IRPs will be purified from a selected, broadly-expressing ExPEC strain and incorporated into nanolipoprotein particles or derivatives of chitosan nanoparticles. Mice will be immunized with these nanoparticle formulations to screen for production of antigen-specific secretory IgA, thereby determining which types of nanoparticles stimulate a robust mucosal immune response to IRPs. Aim 2 will proceed to evaluate the protective efficacy of promising vaccine candidates in a clinically relevant mouse model of UTI. Successful completion of the work outlined in this proposal will provide proof of concept for a novel UTI vaccine based on iron receptors packaged in a nanoparticle delivery vehicle, advance our understanding of mucosal immunity, and provide an evidence- based rationale for the development of outer membrane protein subunit vaccines against bacterial pathogens that invade through a mucosal port of entry.